1. Field of the Invention
This invention relates to an immunoassay method and reagent means for determining nonenzymatically glucosylated proteins and protein fragments (e.g., polypeptides, peptides, and amino acids) in a biological fluid such as human serum or plasma. In particular, the present invention concerns a nonradioisotopic, competitive binding, preferably homogeneous, immunoassay method and test kit for such determination. The invention also involves the preparation and use of immunogens which stimulate the production of antibodies which recognize and bind nonenzymatically glucosylated proteins and fragments thereof. The determination of nonenzymatically glucosylated proteins and protein fragments provides a useful index of control of glucose blood levels.
The consequences of diabetes are now known to be much more extensive than simply high extracellular and low intracellular levels of glucose, which can be managed with insulin therapy. A major difficulty in establishing whether there is a relationship between the degree of hyperglycemia and the long-term complications of diabetes has been the lack of a reliable and objective method for assessing diabetic control. Ingelfinger, F. J., New Engl. J. Med. 296:1228(1977). It has been found that hemoglobin (Hb) undergoes nonenzymatic glucosylation in vivo (see FIG. 1 of the drawings). Thee first step of nonenzymatic glucosylation involves the formation of an aldimine (Schiff base) between the aldehyde group of glucose and an .alpha.- or .epsilon.-amino group of a protein. Once formed, the aldimine can either revert back to free glucose and unglucosylated protein or undergo an Amadori rearrangement to a stable ketoamine. This adduct is in equilibrium with its cyclized forms, principally a pyranose ring structure, which are unique to nonenzymatically glucosylated proteins.
The levels of glucosylated Hb (primarily HbA.sub.1c) have been found to correlate well with the levels of blood glucose that an individual has experienced during the preceding weeks to months (the half-life of Hb in the body is about 60 days) and have been used to monitor the management of blood glucose levels that the diabetic patient has maintained during this time. Reports describing the presence of other nonenzymatically glucosylated proteins in diabetes have appeared: serum albumin [Day, J. F., et al, J. Biol. Chem. 254: 595(1979), Dolhofer, R., et al, Diabetes 29:417(1980), and McFarland, K. F., at al, Diabetes 28:1011(1979)], lens crystallins [Stevens. V. J., et al, Proc. Natl. Acad. Sci. USA 75:2918(1978)], lipoproteins [Schleicher, E., et al, FEBS Letters 129:1(1981)], collagen [Rosenberg, H., et al, BBRC 91:498(1979)], and erythrocyte membrane proteins [Miller, J. A., et al, J. Clin, Invest. 65:896(1980)]. In contrast to HbA.sub.1c which is characterized by a glucosylated N-terminal valine residue, nonenzymatically glucosylated proteins as a general class are characterized by a multiplicity of Amadori-rearranged glucose residues attached to available amino groups on the proteins (principally .epsilon.-amino groups on lysyl residues in the proteins). The extent to which total serum protein is nonenzymatically glucosylated has also been found to correlate well with the mean blood glucose levels maintained by the individual [Bunn, H. F., Am. J. Med. 70:325(1981), Kennedy, A. L., et al, Ann. Int. Med. 95:56(1981), and Yue, D. K., et al, Diabetes 29:296(1980)].
2. Description of the Prior Art
A number of different methods have been developed for the detection of HbA.sub.1c. Only three of these procedures are being used clinically: (1) cation-exchange chromatography [e.g. SMC.RTM. glycosylated hemoglobin (Helena Laboratories) and see U.S. Pat. No. 4,238,196] (2) electrophoresis [e.g. Glytrac.RTM. (Corning), see U.S. Pat. No. 4,222,836], and (3) phenylborate affinity chromatography [e.g., Glyco-Gel B, (Pierce Chemical Co.) and see U.S. Pat. No. 4,269,605]. Although these procedures are offered to provide top speed and convenience, they require isolation of red blood cells, preparation of a hemolysate and separation of HbA.sub.1c from Hb prior to detection. In addition, the final separation step in methods (1) and (2) is based on charge differences between Hb and HbA.sub.1c [i.e., glucosylation neutralizes positive charges (.alpha.-amino groups) on Hb making the HbA.sub.1c more negative relative to Hb]. One consequence of separation by charge is that both the unstable aldimine and the stable ketoamine (see FIG. 1) have the same charge. This can lead to test inaccuracies since the aldimine concentrations will vary widely depending on high transient free glucose concentrations and storage conditions of serum samples.
Certain other methods for determining HbA.sub.1c have been described. For example, U.S. Pat. Nos. 4,200,435; 4,255,385 and 4,274,978 concern a spectrophotometric procedure which takes advantage of the spectral changes that occur when 2,3-diphosphoglycerate (2,3-DPG) or inositol hexaphosphate (phytic acid) binds to HbA near the N-terminal amino acid (valine) of the two .beta.-chains. Since the N-terminal valines of the .beta.-chains are glucosylated in HbA.sub.1c, binding of phytic acid is prevented and no associated spectral changes occur. The change in absorbance induced by phytic acid is, therefore, inversely proportional to the percentage of glycosylated hemoglobin. This technique is limited to the determination of HbA.sub.1c since other serum proteins are nonenzymatically glucosylated at amino groups other than the N-terminal amino group (e.g., at lysyl amino groups). Also, it has been reported that the method suffers from interference by fetal hemoglobin (HbF) [Moore, E. G., at al, Diabetes 29, Suppl 2:70A(1980)] and endogenous 2,3-DPG [Walinder, O., et al, Clin. Chem. 28:96(1982)].
U.S. Pat. No. 4,268,270 describes a method for measuring glycosylated hemoglobin by exposing the sample to oxidizing conditions and measuring the resultant aldehydic compounds.
A radioimmunoassay specific for determining HbA.sub.1c is described in U.S. Pat. No. 4,247,533 based on the preparation of an antibody which binds HbA.sub.1c with substantially no cross-reactivity for hemoglobins A.sub.0, A.sub.1a and A.sub.1b. Such specificity is achieved by administering HbA.sub.1c to an animal to stimulate antibody production and screening resulting aniserum for binding to HbA.sub.1c. The resulting antibody was found to be specific for an antigenic determinant comprising the sugar residue and the adjacent amino acid residues.
An object of the present invention is to provide an antibody selective for binding to Amadori-rearranged glucose residues on glucosylated proteins and protein fragments in general so as to enable the determination of total nonenzymatic glucosylation, principally in relation to serum or plasma proteins. Such determination would be highly selective for nonenzymatic glucosylation because of the selectivity of antibody binding reactions and could be adapted to give a variety of readout signals depending on the immunoassay principle that would be applied. The immunoassay determination of nonenzymatically glucosylated proteins in the blood would provide a very useful index of glycemia.